U.S. patent application number 14/912898 was filed with the patent office on 2016-07-21 for method and composition for detection of oncogenic hpv.
The applicant listed for this patent is NOTRE DAME DU LAC. Invention is credited to Daniel L. Miller, Sharon Stack.
Application Number | 20160208346 14/912898 |
Document ID | / |
Family ID | 52484259 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160208346 |
Kind Code |
A1 |
Stack; Sharon ; et
al. |
July 21, 2016 |
METHOD AND COMPOSITION FOR DETECTION OF ONCOGENIC HPV
Abstract
Probes for the detection of oral oncogenic human papillomavirus
(HPV) are described. The probe includes a polynucleotide having at
least 90% sequence identity to a polynucleotide complementary to a
microRNA that has altered expression in response to oncogenic HPV
infection. A method for detecting oncogenic HPV in a subject is
also described. The method comprises the steps of (A) providing a
sample from a subject; (B) measuring the expression level of a
microRNA having altered expression in response to oncogenic HPV
infection using a probe; and (C) determining that the subject is
infected by an oncogenic HPV if the expression level is increased
or decreased in comparison with a control.
Inventors: |
Stack; Sharon; (Notre Dame,
IN) ; Miller; Daniel L.; (Notre Dame, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOTRE DAME DU LAC |
Notre Dame |
IN |
US |
|
|
Family ID: |
52484259 |
Appl. No.: |
14/912898 |
Filed: |
August 19, 2014 |
PCT Filed: |
August 19, 2014 |
PCT NO: |
PCT/US14/51692 |
371 Date: |
February 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61867380 |
Aug 19, 2013 |
|
|
|
61942485 |
Feb 20, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/708 20130101; C12Q 2600/178 20130101 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The present invention was made with government support under
NIH-NIDCR F31DE021926-02 awarded by the National Institutes of
Health-National Institute of Dental and Craniofacial Research
(NIH-NIDCR). This invention was also made with government support
under NIH-NCI RO1 CA085870 awarded by the National Institutes of
Health-National Cancer Institute (NIH-NCI). The US government has
certain rights in this invention.
Claims
1. A probe for detecting oncogenic Human papillomavirus (HPV),
comprising a polynucleotide that has at least 90% sequence identity
to a polynucleotide complementary to at least one microRNA that has
altered expression in response to oncogenic HPV infection.
2. The probe of claim 1, wherein expression of the microRNA is
up-regulated in response to oncogenic HPV infection.
3. The probe of claim 1, wherein expression of the microRNA is
down-regulated in response to oncogenic HPV infection.
4. The probe of claim 2, wherein the up-regulated microRNA is
selected from the group consisting of SEQ ID No:1, SEQ ID No:2, SEQ
ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID
No:8, and SEQ ID No:9.
5. The probe of claim 4, wherein the polynucleotide that that has
at least 90% sequence identity to a polynucleotide complementary to
the up-regulated microRNA is selected from the group consisting of
SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID
No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, and SEQ ID
No:22.
6. The probe of claim 3, wherein the down-regulated microRNA is
selected from the group consisting of SEQ ID No:10, SEQ ID No:11,
SEQ ID No:12, and SEQ ID No:13.
7. The probe of claim 6, wherein the polynucleotide that that has
at least 90% sequence identity to a polynucleotide complementary to
the down-regulated microRNA is selected from the group consisting
of SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, and SEQ ID No:26.
8. A method for determining if a subject is infected by an
oncogenic HPV, comprising: obtaining a sample from the subject;
determining the level of a microRNA whose expression is altered in
response to infection by an oncogenic HPV, comparing the level of
the microRNA to a control level, and determining that the subject
is infected by an oncogenic HPV if the level of the microRNA is
altered relative to that of the control level.
9. The method of claim 8, wherein the microRNA is up-regulated in
response to oncogenic HPV infection.
10. The method of claim 8, wherein the microRNA is down-regulated
in response to oncogenic HPV infection.
11. The method of claim 9, wherein the up-regulated microRNA is
selected from the group consisting of SEQ ID No:1, SEQ ID No:2, SEQ
ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID
No:8, and SEQ ID No:9.
12. The method of claim 11, wherein a probe complementary to the
up-regulated microRNA, is selected from the group consisting of SEQ
ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18,
SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, and SEQ ID No:22.
13. The method of claim 10, wherein the down-regulated microRNA is
selected from the group consisting of SEQ ID No:10, SEQ ID No:11,
SEQ ID No:12, and SEQ ID No:13.
14. The method of claim 13, wherein a probe complementary to the
down-regulated microRNA is selected from the group consisting of
SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, and SEQ ID No:26.
15. The method of claim 8, wherein the level of the microRNA is
determined using an assay selected from the group consisting of
RT-PCR, Fluorescence In Situ Hybridization and use of a
microfluidic chip.
16. The method of claim 15, wherein the level of the microRNA is
determined using an RT-PCR assay.
17. The method of claim 15, wherein the level of microRNA is
determined using Fluorescence In Situ Hybridization using a probe
selected from the group consisting of SEQ ID No:14, SEQ ID No:15,
SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID
No:20, SEQ ID No:21, and SEQ ID No:22.
18. The method of claim 15, wherein the level of microRNA is
determined using a microfluidic chip and a probe selected from the
group consisting of SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ
ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21,
and SEQ ID No:22.
19. The method of claim 8, wherein the sample is an oral rinse.
20. The method of claim 19, wherein the oral rinse has a volume of
at least 10 mL.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/867,380, filed Aug. 19, 2013, and U.S.
Provisional Application No. 61/942,485, filed Feb. 20, 2014, both
of which are incorporated herein by reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 17, 2014, is named UND-023707 Sequence_ST25 and is 5100
bytes in size.
BACKGROUND
[0004] 1. Field of the Invention
[0005] The present disclosure generally relates to methods of
disease detection and prognosis and, more particularly, to methods
for detecting oncogenic viruses, such as oral oncogenic HPV.
[0006] 2. Description of Related Art
[0007] There are various techniques for detecting an infection of
the head and neck. While the overall incidence of head and neck
squamous cell carcinoma (HNSCC) has decreased since 1988, the
incidence of tumors localized in the oropharynx has shown a
striking 225% increase. With regards to head and neck cancer
detection, current methods identify thyroid cancer, head and neck
squamous cell carcinoma, or the relationship between two diseases
in the head and neck, to name a few. See Miller et al., Biochem J.
443, 339-353 (2012). However, the detection of these diseases does
not address the rising number of HPV-related cancers, particularly
in the mouth.
[0008] Recently, the percentage of HPV-related diseases has been
increasing. The ability to distinguish oncogenic human
papillomavirus (HPV) versus non-oncogenic HPV is very important
regarding the consequences to a patient. The presence of oncogenic
HPV versus non-oncogenic HPV determines the further treatment of
the patient. The current techniques to distinguish HPV-related
cancer require two different sets of tests: one to determine HPV+
or HPV-, and an additional test is required to determine if the
sample is oncogenic or not oncongenic. None of these techniques
alone were particularly well suited to provide a sensitive,
specific, and non-invasive way of differentiating oncogenic viruses
from non-oncogenic infections, wherein the percentage of accuracy
is relatively high. The optimal method to detect oral oncogenic HPV
remains unaddressed by current techniques.
[0009] With regards to HPV detection, current methods provide
cervical cancer diagnostic kits and therapies. However, these
methods, among other things, do not address the population of those
infected with oncogenic HPV, which can have substantially different
infections. Furthermore, these methods require examination of
samples that are taken invasively, by methods such as obtaining a
blood sample or pap smear. Additionally, these methods typically
involve the analysis of molecules like proteins or DNA, which have
high false-positive detection rates.
[0010] What is not available is a test for distinguishing oncogenic
viruses from non-oncogenic viruses that is more specific than
proteins or DNA, using a sample that is obtained in a non-invasive
way from patients. Such a test would provide comfort to the patient
and a higher accuracy of detection.
[0011] Currently, clinicians use microRNAs to detect stomach
cancers, pancreatic cancers, and cervical cancer. These methods
cannot be made analogous to diseases of the head and neck, and
certainly cannot address an HPV-related cancer in the mouth because
of the genotypic differences in these variant diseases. None of
these detection techniques provide a sensitive test for a
non-invasively obtained sample. Accordingly, there is therefore a
need for methods to more sensitively detect oncogenic HPV.
SUMMARY OF THE INVENTION
[0012] High-risk human papillomavirus (HPV) is a causative agent
for an increasing subset of oropharyngeal squamous cell carcinomas
(OPSCC) and current evidence supports these tumors as having
identifiable risk factors and improved response to therapy. However
the biochemical and molecular alterations underlying the
pathobiology of HPV-associated OPSCC (designated HPV+OPSCC) remain
unclear. Herein we profile microRNA (miRNA) expression patterns in
HPV+OPSCC to provide a more detailed understanding of pathological
molecular events and to identify biomarkers that may have
applicability for early diagnosis, improved staging, and prognostic
stratification. Differentially expressed miRNAs were identified in
RNA isolated from an initial clinical cohort of HPV+/-OPSCC tumors
by qPCR-based miRNA profiling. This oncogenic miRNA panel was
validated using miRNAseq and clinical data from The Cancer Genome
Atlas (TCGA) and miRNA-in situ hybridization (miR-ISH). The
HPV-associated oncogenic miRNA panel has potential utility in
diagnosis and disease stratification as well as in mechanistic
elucidation of molecular factors that contribute to OPSCC
development, progression and differential response to therapy.
[0013] The shortcomings of the related art are overcome and
additional advantages are provided through the provision of probes
for the detection of oncogenic HPV. The probes are polynucleotide
complementary to a microRNA having altered expression in response
to an oncogenic HPV infection, and include probes having 90% or
more sequence identity to such probes.
[0014] Additional shortcomings of the related art are overcome and
additional advantages are provided through a method for detection
of oncogenic HPV comprising providing a sample of oral rinse from a
subject, measuring the expression level of microRNA having an
altered level of expression in response to oncogenic HPV infection
using a complementary probe, wherein the probe has at least 90%
sequence identity to a directly complementary probe, and
determining the presence of oncogenic HPV.
[0015] Additional features and advantages are realized through the
techniques of the invention described herein. Other embodiments and
aspects are described in detail herein and are considered a part of
the claimed technology. For a better understanding of the
advantages and features of the technology, refer to the description
and the drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The present invention may be more readily understood by
reference to the following figures, wherein:
[0017] FIG. 1 provides a graph illustrating an example of small RNA
expression across 13 samples. Micro- and Small nucleolar RNAs were
used as positive controls to assess the quality of RNA extracted
from Formalin-Fixed, Paraffin-Embedded (FFPE) tissues blocks that
were obtained between the years 2006-2011. Because these
universally expressed ncRNA were stable across samples, it was
concluded that miRNA is relatively stable and protected in
paraffin;
[0018] FIG. 2 provides a bar graph illustrating the p-values from
miR-320a, miR-222-3p, and miR-93-5p based on the data shown in
Table 2;
[0019] FIG. 3 provides a bar graph illustrating the p-values from
miR-451a, miR-199a-3p//145-5p, miR-143-3p, miR-126-5p, and
miR-126-3p based on the data shown in Table 3;
[0020] FIG. 4 provides a bar graph illustrating that miRs
differentially expressed in 2 HPV+vs 2HPV- cell lines;
[0021] FIG. 5 illustrates levels of miR-145 repressed in
HPV-31-positive organotypic rafts. (A) is an example of qPCR
analysis of miR-145 levels in 13-day-old organotypic raft cultures
of matched normal keratinocytes (viv) and HPV-positive cells that
stably maintain episomes (viv-31gen). The data are represented as
fold changes with respect to miR-145 levels. (B and C) are examples
of luciferase reporter assays measuring responsiveness to miR-145
of sequences in HPV-31 E1 and E2. (D) is an example of mutation of
miR-145 seed and are averages with standard errors from three
independent experiments. (E) is an example of a schematic
representation of HPV- 31 genome with miR-145 target sequences
indicated;
[0022] FIG. 6 illustrates an example of levels of miR-145 decrease
upon differentiation of HPV-31-positive cells. (A) is an example of
E7 protein mediated repression of miR-145. The data are from qPCR
and are normalized to U6 levels and are represented as fold
difference from miR-145 levels in normal keratinocytes. (B) is an
example of qPCR analysis of miR-145 levels in normal keratinocytes
induced to differentiate in high calcium media. The data are
normalized to U6 levels and are represented as fold change relative
to miR-145 levels in undifferentiated cultures. (C) is an example
of qPCR analysis of miR-145 levels in cells stably maintaining
HPV-31 episome monolayer cultures upon differentiation in high
calcium. The data are normalized to U6 levels and are represented
as fold change from levels seen in undifferentiated cells;
[0023] FIG. 7 illustrates an example of high-level expression of
miR-145 from heterologous expression vectors blocks HPV genome
amplification, late gene expression, and induction of KLF-4. (A) is
an example of a southern blot analysis of supercoiled episomal
viral DNA levels upon differentiation of CIN-612 cells with forced
expression of miR-145. Averages from three independent experiments
with standard errors are shown in the bar graph. UD,
undifferentiated. (B) is an example of a southern blot analysis of
supercoiled episomal viral DNA levels following differentiation of
CIN-612 cells expressing high levels of miR-146a (CIN-612 miR-146a
cells), vector control, and mock-transfected cells. Quantification
of the band intensities is shown in the bar graph. (C) is an
example of a northern blot analysis of early and late viral
transcripts during differentiation of CIN-612 miR-145 cells and
mock-infected control cells. Arrows indicate early transcripts
encoding E6*, E7, E1, E2, E5, E6*, E7, and E1E4, as well as late
transcripts encoding E1E4 and E5. Results from three independent
experiments with standard errors are shown. (D) is an example of a
western blot analysis showing levels of KLF-4 and Oct-4 in cell
extracts from raft cultures. A nonspecific band (at 60 kDa) was
detected in CIN-612 cultures. The individual bands were quantified
and normalized are represented as fold differences under each
band.
[0024] FIG. 8 illustrates a scatter plot and graphical analysis of
an example of a microarray analysis of microRNA expression showing
HPV `over-expressed` microRNA data from cell lines obtained using
an exiqon microarray;
[0025] FIG. 9 illustrates a graphical example of a microarray
analysis of relative microRNA expression through fold change
showing HPV `over-expressed` microRNA data from cell lines obtained
using an exiqon microarray;
[0026] FIG. 10 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for miR-320a,
miR-93, and miR-222-3p;
[0027] FIG. 11 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for miR-106a,
miR-15a, and miR-141-;
[0028] FIG. 12 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for miR-200c,
miR-335, and miR-26b;
[0029] FIG. 13 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for miR-33a;
[0030] FIG. 14 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for miR-26b;
[0031] FIG. 15 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for miR-34a,
miR-145-5p, miR-143-3p and miR-451; and
[0032] FIG. 16 illustrates an example of analysis on raw patient
data from FFPE samples according to each patient for
miR-199a-3p//199b-3p, miR-126-3p, miR-199b-5p, and miR-126-5p.
[0033] FIG. 17 shows the HPV prevalence in FFPE cases. (A) Clinical
history for patients diagnosed between 2006-2011 was reviewed,
archived FFPE tissue blocks were assessed for available tissue, and
available H&E slides reviewed. Following an IRB approved
protocol, tissues were sectioned, stained for p16 protein
expression and scored as positive or negative as described. Results
show that 58% of cases are HPV+. (B) The average ages in the p16+
and p16- cohorts under study were 56.49 and 61.00 years of age,
respectively.
[0034] FIG. 18 shows the profiling of miRNA expression on FFPE
samples by q-rt-PCR. (A) Tumors from 23 patients, including fifteen
p16+ and eight p16- samples were profiled as described. Panel shows
a unsupervised hierarchical clustering heat map of normalized data
(prior to non-specific filtering or testing) representing 511
miRNAs. The dendogram at the top of the heat map illustrates which
patient samples have the most similar miRNA profile, while the
dendrogram on the left y-axis illustrates which miRNAs have similar
profiles across patients. Items which are most similar are linked
sooner to each other than items which are less similar. The panel
at the bottom provides clinical information for each sample, with a
black square marking the presence of the indicated variable (gray
indicates missing data); green (8/10 HPV+) and salmon (6/13 HPV-)
shading indicate how the samples cluster into two groups. (B)
Significantly up- or down-regulated miRNAs (p<0.01). (C)
Unsupervised hierarchical clustering heatmap based on 43 selected
miRNAs. The dendogram (top) is broken into 6 distinct groups with
the majority of the samples falling into 3 groups: mostly smokers
regardless of HPV status; all HPV+ non-smokers, and mixed.
[0035] FIG. 19 shows the analysis of TCGA Cohort 1 miRNAseq data.
(A) Patients comprising TCGA Cohort 1 were identified as described.
Graph shows comparison of the 7 differentially expressed miRNAs
identified by PCR profiling of microdissected FFPE (fold changes in
log 2 scale for symmetry) versus results obtained from analysis of
TCGA Cohort 1 miRNA-seq data. A strong concordance between the
datasets was obtained based on Spearmans rank correlation
(Rho=0.85, p=0.02) and Pearson's product-moment correlation
(r=0.83, p=0.02; 95% CI 0.21-0.97). A best-fit line indicates this
relative concordance, while a 45-degree reference line (dotted)
indicates that there is not perfect absolute agreement between the
data sets and assay technique. (B) Mean normalized miRNA read
counts showing miR-199a as a validated HPV-associated miRNA
(p=9.8E-03). (C) miR-106b is expressed from the same primary
polycistronic transcript as miR-93 (FIG. 2) and shares the same
functionally relevant nucleotide (seed) sequence. Mean normalized
miRNA read count shows upregulation of this paralog in HPV+ tumors
(p=1.6E-04). (D) Mean normalized read counts showing miR-9 is
significantly upregulated in HPV+(p=5.4E-04). This miRNA was
upregulated, but not significantly in FFPE qPCR experiments (FIG.
2), and has been identified by two independent published studies as
an HPV-associated miRNA in OPSCC [21, 27]. All bars indicate 95%
confidence limits, not standard error of the mean. (E) Heatmap of
normalized expression data with color scale representing greater or
lesser levels of relative expression across TCGA HNSCC samples
(columns) and miRs (rows). Unsupervised hierarchical clustering of
both the samples and the miRs was carried out using Euclidean
distance and the average linkage methods, and the resulting
dendrograms are shown in the margins, where `+` or `-` indicates
HPV status. Items that are most similar cluster lower in the
dendrogram. There appear to be 2 distinct clusters of samples, one
entirely HPV+ and the other mostly HPV-, as well as 4 distinct
clusters of miRNAs. As an example reflecting the color scale, the
bottom rows show miRNA expressed across all samples at levels
between 15,000-320,000 read per million miRNA mapped.
[0036] FIG. 20 shows the analysis of TCGA Cohort 2 miRNAseq data.
(A) Patients comprising TCGA Cohort 2 were identified as described.
Graph shows comparison of the 7 differentially expressed miRNAs
identified by PCR profiling of microdissected FFPE (fold changes in
log 2 scale) versus results obtained from analysis of TCGA Cohort 2
miRNA-seq data. A strong concordance between datasets was obtained
based on Spearmans rank correlation (Rho=0.75, p=0.06) and
Pearson's product-moment correlation (r=0.78, p=0.03; 95% CI
0.07-0.96). A best-fit line indicates this relative concordance,
while a 45-degree reference line (dotted) indicates that there is
not perfect absolute agreement between the data sets and assay
technique. (B) Mean normalized miRNA read counts showing miR-106b
as a validated HPVassociated miRNA (p=5.4E-04). (C) Mean normalized
miRNA read counts showing miR-9 as a validated HPV-associated miRNA
(p=5.8E-04). All bars indicate 95% confidence limits, not standard
error of the mean.
[0037] FIG. 21 shows the In-situ hybridization analysis of miR-9
expression in HNSCC. (A-D) Low power images depicting ISH patterns
for miR-9 in four OPSCC tissue cores. The ISH signal is represented
by a deep shade. The counter stain for miRNA-probed tissues
sections was nuclear fast red; therefore, darker coloration
represents ISH signal and lighter is the counterstain. (A,C) HPV+
tumors, (B,D) HPVtumors. HPV+ tumors show strong ISH signal while
HPV- tumors have weak or absent signal. Magnification 100.times..
(E-H) High power images depicting ISH patterns for miR-9 in four
OPSCC tissue cores. Staining in HPV+ cores is punctate (E) or
diffuse (G) while HPV- tumors have weak or absent signal.
Magnification 400.times.. (I) Odds ratios and mosaic plots of high
tumor miR-9 occurring in the setting of p16+ disease. The odds that
high-tumor miR-9 expression occurs in the setting of p16+ disease
are more than 3 times greater (OR=3.38; p<0.001; 95% CI
1.84-6.26) than the odds of low-tumor miR-9 expression; similarly,
the odds of diffuse miR-9 ISH are nearly four times greater
(OR=3.87; p<0.001; 95% CI 2.10-7.20) than the odds of
non-diffuse miR-9 ISH.(J) The odds ratios increase when the outcome
is HPV mRNA ISH. The model prediction based on HPV mRNA ISH is
associated has sensitivity=0.62 and specificity=0.875.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present disclosure provides a method and composition
that utilizes host-specific macromolecules, such as microRNAs, to
detect the presence of oncogenic infections that previously were
unable to be distinguished so specifically, without a high
false-positive rate.
[0039] The following description of example methods and composition
is not intended to limit the scope of the description to the
precise form or form detailed herein. Instead the following
description is intended to be illustrative so that others may
follow its teachings.
[0040] As used herein, a subject can be a vertebrate, more
specifically a mammal (e.g., a human, horse, cat, dog, cow, pig,
sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles,
amphibians, fish, and any other animal. The term does not denote a
particular age. Thus, adult, juvenile, and newborn subjects are
intended to be covered. As used herein, patient or subject may be
used interchangeably and can refer to a subject afflicted with a
disease or disorder (e.g. oncogenic HPV). The term patient or
subject includes human and veterinary subjects.
[0041] The term "control" refers to an experiment or observation
used to minimize the effects of variables. Through the comparison
of a control measurement and a measurement, reliability can be
increased. In one embodiment, a dataset may be obtained from
samples from a group of subjects known to have a particular
oncogenic infection. The expression data of the biomarkers in the
dataset can be used to create a control value that is used in
testing samples from new subjects. In such an embodiment, the
control is a predetermined value for each biomarker or set of
biomarkers obtained from subjects with oncogenic infection.
[0042] The term "polynucleotide" as used herein refers to a nucleic
acid sequence including DNA, RNA, and microRNA and can refer to
markers which are either double stranded or single stranded.
Polynucleotide can also refer to synthetic variants with
alternative sugars like the LNA.
[0043] The term "complementary" as used herein refers to nucleotide
sequences that complement the polynucleotides' reverse sequence.
Complementarity is the base principle of DNA replication and
transcription as it is a property shared between two DNA or RNA
sequences, such that when they are aligned antiparallel to each
other, the nucleotide bases at each position in the sequences will
be complementary. Complementarity is achieved by distinct
interactions between nucleobases: adenine, thymine (uracil in RNA),
guanine and cytosine. Adenine (A) and guanine (G) are purines,
while thymine (T), cytosine (C) and uracil (U) are pyrimidines.
Purines are larger than pyrimidines. Both types of molecules
complement each other and can only base pair with the opposing type
of nucleobase. In nucleic acid, nucleobases are held together by
hydrogen bonding, which only works efficiently between adenine and
thymine and between guanine and cytosine. The base complement A=T
shares two hydrogen bonds, while the base pair GC has three
hydrogen bonds.
[0044] The degree of complementarity between two nucleic acid
strands may vary, from complete complementarity (each nucleotide is
across from its opposite) to no complementary (each nucleotide is
not across from its opposite) and determines the stability of the
sequences to be together. Lesser degrees of complementarity are
referred to herein by percentages of sequence identity as compared
with a sequence having 100% complementarity. Embodiments of the
invention include sequences having at least about 70% to at least
about 100% sequence identify to a complementary sequence. For
example, probes can have sequences having at least 70%, 75%, 80%,
85%, 90%, 95%, or 100% sequence identify with a complementary
probe. In another example, the sequence identity can be at least
about 80% to at least about 95% that of a complementary sequence.
In a preferred embodiment, the probe can have at least about 87%,
88%, 89%, 90%, 91%, or 92% sequence identity to a complementary
probe. In another preferred embodiment, the probe can have at least
90% sequence identity to a complementary probe.
[0045] The term "microRNAs" as used herein refers to a class of
small RNAs typically between 15 and 30 nucleotides long. microRNAs
can refer to a class of small RNAs that play a role in gene
regulation. In a preferred embodiment, a microRNA refers to a
human, small RNA of 20, 21, 22, 23, 24, 25, or 26 nucleotides
long.
[0046] The term "expression" as used herein refers to the presence
of biomarkers, such as the presence of microRNAs. A "change in
expression" refers to a difference in the measurement of the
biomarkers in a sample and known, controlled measurements of the
biomarkers, such as the difference in level of microRNAs expressed
of the biomarkers.
[0047] The term "HPV" as used herein refers to Human
papillomavirus. HPV is a DNA virus from the papillomavirus family
that is capable of infecting humans. An oncogenic HPV is an HPV
that has a relatively high propensity, in comparison with other
types of HPV, to induce the development of cancer over time. The
oncogenic potential of human papillovaviruses are known to those
skilled in the art. See Madkan et al., British Journal of
Dermatology, 157(2):228-241 (2007). Examples of HPV subtypes that
turn on oncogenic gene expression include subtypes HPV16 and
HPV18.
[0048] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic or physiologic
effect. The effect may be therapeutic in terms of a partial or
complete cure for a disease or an adverse effect attributable to
the disease. "Treatment," as used herein, covers any treatment of a
disease in a mammal, particularly in a human, and can include
inhibiting the disease or condition, i.e., arresting its
development; and relieving the disease, i.e., causing regression of
the disease.
[0049] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0050] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0051] As used herein and in the appended claims, the singular
forms "a", "and", and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a sample" also includes a plurality of such samples and reference
to "the microRNA" includes reference to one or more microRNA, and
so forth.
[0052] The term "marker" as used herein refers to a microRNA used
as an indicator of a biological state or condition, the condition
being infection with oncogenic HPV. Changes in the level of
expression of microRNA (SEQ ID No.'s 1-13) are associated with
oncogenic HPV. SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4,
SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, and SEQ ID
No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No12, and SEQ ID No:13.
The SEQ IDs of the microRNA markers useful for indicating the
presence of oral oncogenic HPV are defined as in Tablet. The
microRNA can be detected or measured by an analytic device such as
a kit or a conventional laboratory apparatus, which can be either
portable or stationary.
TABLE-US-00001 TABLE 1 HPV-associated miRNA and probes Probe SEQ
SEQ ID miR Probe ID NO. Sequence Name Sequence NO. 1 ucuuugguuauc
miR-9- ucauacagcuag 14 uagcuguauga 5p auaaccaaaga 2 caaagugcucau
miR-20b- cuaccugcacu 15 agugcagguag 5p augagcauuug 3 cauugcacuugu
miR-25- ucagaccgaga 16 cucggucuga 3p caagugcaaug 4 caaagugcuguu
miR-93- cuaccugcacga 17 cgugcagguag 5p acagcacuuug 5 uaaagugcugac
miR- aucugcacug 18 agugcagau 106b-5p cagcacuuua 6 agcuacaucugg miR-
acccaguagcc 19 cuacugggu 222-3p agauguagcu 7 aaaagcuggguu miR-320a
ucgcccucuca 20 gagagggcga acccagcuuuu 8 cgcauccccuag miR-324-
acaccaaugccc 21 ggcauuggugu 5p uaggggaugcg 9 aauugcacggua miR-363-
uacagauggau 22 uccaucugua 3p accgugcaauu 10 ucguaccgugag miR-126-
cgcauuauuac 23 uaauaaugcg 3p ucacgguacga 11 ugagaugaagca miR-143-
gagcuacagug 24 cuguagcuc 3p cuucaucuca 12 ugagaugaagca miR-145-
gagcuacagug 25 cuguagcuc 3p cuucaucuca 13 acaguagucugc miR-100a-
uaaccaaugug 26 acauugguua 3p//miR- cagacuacugu 199b-3p
[0053] One aspect of the invention is directed to polynucleotide
probes. The term "probe" as used herein refers to a polynucleotide
sequence that will hybridize to a complementary target sequence. In
one example, the probe hybridizes to a microRNA sequence. The
probes provided herein have nucleotide sequences that have 90%
sequence identity to polynucleotide sequences that are the
complement of a microRNA having altered expression as a result of
HPV infection. These probes (exemplified by SEQ ID No.'s 14-26) can
detect microRNA markers. Thus, because SEQ ID No.'s 1-13 are
markers of oral oncogenic HPV, the corresponding probes (SEQ ID
No.'s 14-26) can be used to detect these markers. A method of
detecting HPV or oncogenic HPV can use at least one, at least two,
at least three, at least four, at least five, at least six, at
least seven, at least eight, at least nine, at least ten, at least
eleven, at least twelve, or at least thirteen probes selected from
the group consisting of SEQ ID No: 14, SEQ ID No:15, SEQ ID No:17,
SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID
No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, and SEQ ID No:26.
In a preferred embodiment, at least four probes are selected from
the group consisting of SEQ ID No: 14, SEQ ID No:15, SEQ ID No:17,
SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID
No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, and SEQ ID No:26.
The probes and their associated SEQ IDs are provided in Tablet.
[0054] Another aspect of the invention provides a method for
determining if a subject is infected by an oncogenic HPV that
includes the steps of obtaining a sample from the subject;
determining the level of a microRNA whose expression is altered in
response to infection by an oncogenic HPV, comparing the level of
the microRNA to a control level, and determining that the subject
is infected by an oncogenic HPV if the level of the microRNA is
altered relative to that of the control level. To determine if
increased or decreased expression of the microRNA has occurred, the
level of microRNA is compared to a control level.
[0055] The degree of an increase in the expression level of the
present microRNA when determined as indicating the presence of
oncogenic HPV can be, for example, preferably 50% or more, more
preferably 75% or more, still more preferably 100% or more as a
percentage relative to a control, and the degree of a decrease in
the expression level of the present microRNA when determined as
indicating the presence of oncogenic HPV can be, for example,
preferably 25% or more, more preferably 50% or more, still more
preferably 75% or more as a percentage relative to a control.
[0056] A number of the methods described herein include the step of
obtaining a biological sample from the subject. A "biological
sample," as used herein, is meant to include any biological sample
from a subject that is suitable for analysis for detection of the
microRNA whose level varies in response to infection of the subject
by oncogenic HPV. Suitable biological samples include but are not
limited to bodily fluids such as blood-related samples (e.g., whole
blood, serum, plasma, and other blood-derived samples), urine,
sputem, cerebral spinal fluid, bronchoalveolar lavage, and the
like. Another example of a biological sample is a tissue sample.
The probes can be used to detect oncogenic HPV using samples
obtained from a variety of tissue sites. In some embodiments
samples are obtained from anal, cervical, and penile tissue. The
level of microRNA can be assessed either quantitatively or
qualitatively, and detection can be determined either in vitro or
ex vivo.
[0057] A biological sample may be fresh or stored. Biological
samples may be or have been stored or banked under suitable tissue
storage conditions. The biological sample may be a tissue sample
expressly obtained for the assays of this invention or a tissue
sample obtained for another purpose which can be subsampled for the
assays of this invention. Preferably, biological samples are either
chilled or frozen shortly after collection if they are being stored
to prevent deterioration of the sample.
[0058] In some embodiments, the sample is an oral rinse sample. The
oral rinse can be a liquid. The liquid can be, but is not limited
to any of the following: mouthwash, saline rinse, liquid rinse and
liquid mixture. In one embodiment, at least about 8 to at least
about 35 mL of oral rinse is used. In another embodiment, 10 to 30
mL is used. For example, a volume of 10, 15, 20, 25, or 30 mL is
used. In another example, a volume of at least about 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, or 35 mL. In a preferred embodiment, volume
is 10 to 20 mL oral rinse. In another preferred embodiment, volume
is 13 to 18 mL. This preferred embodiment provides an optimal
balance between comfort to the subject while allowing maximum
access to the cells at the rinsing and gargling step.
[0059] To obtain an oral sample, the oral rinse is swished and/or
gargled in a subject's mouth for less than one minute (e.g., 10 to
30 seconds) before being expelled. Swishing is the process of
holding an oral rinse in the mouth while moving it using the cheeks
and tongue, while gargling is the process of washing one's mouth
and throat with a liquid kept in motion by exhaling through it. For
example, the oral sample can be swished and/or gargled 10, 15, 20,
25, or 30 seconds. In another example, the oral sample can be
swished and/or gargled 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35 seconds. In a preferred embodiment, the oral sample is swished
and/or gargled is 23 to 33 seconds. This preferred embodiment
provides the advantage of optimal time of swishing and gargling in
the mouth to obtain the maximum number of cells for detection
analysis. In an alternate embodiment, the sample could be taken as
a cervical sample. In an additional alternate embodiment, the
sample could be taken as an anal sample.
[0060] A person skilled in the art will appreciate that a number of
methods can be used to detect or quantify the level of microRNA
biomarkers within a sample, including microarrays, PCR (including
quantitative RT-PCR), nuclease protection assays, in situ
hybridization, and microfluidics devices. In one embodiment, the
assay used is PCR, as described in Example 3 which uses
quantitative RT-PCR.
[0061] The microarray method is not particularly limited provided
that it can measure the level of the microRNA whose expression
changes in response to infection by oncogenic HPV; examples thereof
can include a method which involves labeling the RNA extracted from
a tissue with a label (preferably a fluorescent label), contacting
the RNA with a microarray to which a probe consisting of a
polynucleotide (preferably DNA) consisting of a nucleic acid
sequence complementary to the microRNA to be identified or a part
thereof is fixed for hybridization, washing the microarray, and
measuring the expression level of the remaining microRNAs on the
microarray.
[0062] The type of the nucleotide of the nucleic acid sequence is
not particularly limited provided that it can specifically
hybridize to the microRNA of the present invention. The length of
the part of the polynucleotide is not particularly limited provided
that it specifically hybridizes to the predetermined microRNA
according to the present invention; however, it is preferably 10 to
100 mers, more preferably 10 to 40 mers in view of securing the
stability of hybridization. The polynucleotide or a part thereof
can be obtained by chemical synthesis or the like using a method
well known in the art.
[0063] The array to which the polynucleotide or a part thereof is
fixed is not particularly limited; however, preferred examples
thereof can include a glass substrate and a silicon substrate, and
the glass substrate can be preferably exemplified. A method for
fixing the polynucleotide or a part thereof to the array is not
particularly limited; a well-known method may be used.
[0064] The quantitative PCR method is not particularly limited
provided that it is a method using a primer set capable of
amplifying the sequence of the microRNA and can measure the
expression level of the present microRNA; conventional quantitative
PCR methods such as an agarose electrophoresis method, an SYBR
green method, and a fluorescent probe method may be used. However,
the fluorescent probe method is most preferable in terms of the
accuracy and reliability of quantitative determination.
[0065] The primer set for the quantitative PCR method means a
combination of primers (polynucleotides) capable of amplifying the
sequence of the microRNA. The primers are not particularly limited
provided that they can amplify the sequence of the microRNA;
examples thereof can include a primer set consisting of a primer
consisting of the sequence of a 5' portion of the sequence of a
microRNA of the present invention (forward primer) and a primer
consisting of a sequence complementary to the sequence of a 3'
portion of the microRNA (reverse primer). Here, the 5' means 5' to
the sequence corresponding to the reverse primer when both primers
were positionally compared in the sequence of a mature microRNA;
the 3' means 3' to the sequence corresponding to the forward primer
when both primers were positionally compared in the sequence of a
microRNA.
[0066] Preferred examples of the 5' sequence of a microRNA can
include a sequence 5' to the central nucleic acid of the microRNA
sequence; preferred examples of the 3' sequence of the microRNA can
include a sequence 3' to the central nucleic acid of the microRNA
sequence. The length of each primer is not particularly limited
provided that it enables the amplification of the microRNA;
however, each primer is preferably a 7-to-10-mer polynucleotide.
The type of the nucleotide of a polynucleotide as the primer is
preferably DNA because of its high stability.
[0067] In some embodiments, a fluorescent probe is used. The
fluorescent probe is not particularly limited provided that it
comprises a polynucleotide consisting of a nucleic acid sequence
complementary to the sequence of the present microRNA or a part
thereof; preferred examples thereof can include a fluorescent probe
capable of being used for the TaqMan.TM. probe method or the
cycling probe method; the fluorescent probe capable of being used
for the TaqMan.TM. probe method can be particularly preferably
exemplified. Examples of the fluorescent probe capable of being
used for the TaqMan.TM. probe method or the cycling probe method
can include a fluorescent probe in which a fluorochrome is labeled
5' thereof and a quencher is labeled on 3' thereof. The
fluorochrome, quencher, donor dye, acceptor dye used or the like
used with a fluorescent probe are commercially available.
[0068] In some embodiments, the level of microRNA can be determined
using a microfluidic chip. Use of a microfluidic chip includes the
steps of making an assay mixture containing at least one microRNA;
providing a microchamber electrochemical cell comprising a
substrate defining a pair of opposing microchannels; at least one
ion exchanging nanomembrane coupled between the opposing
microchannels such that the microchannels are connected to each
other only through the nanomembrane; wherein said at least one
polynucleotide is selected from the group consisting of SEQ ID
No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ
ID No:19, SEQ ID No:20, SEQ ID No:21, and SEQ ID No:22; a device
for measuring the electrical current of potential across the
nanomembrane; flowing the assay mixture through the opposing
microchannels; and detecting a change in the measure electrical
current of potential across the nanomembrane to qualify the
presence of the microRNA.
[0069] Conventional techniques of molecular biology, microbiology
and recombinant DNA techniques, are within the skill of the art.
Such techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J.
Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes & S.
J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B.
Perbal, 1984); and a series, Methods in Enzymology (Academic Press,
Inc.); Short Protocols In Molecular Biology, (Ausubel et al., ed.,
1995).
[0070] A person skilled in the art will appreciate that a number of
detection agents can be used to determine the expression of the
biomarkers. For example, to detect microRNA biomarkers, probes,
primers, complementary polynucleotide sequences or polynucleotide
sequences that hybridize to the microRNA products can be used. In
some embodiments, reverse complementary poylynucleotides serve as
probes for microRNA markers. In alternate embodiments, a
complementary polynucleotide sequence that hybridizes to the target
polynucleotide sequence can be used to detect expression of the
microRNA markers.
[0071] Another aspect of the invention provides a method of
treating oncogenic HPV infection in a subject in need thereof that
includes the steps of obtaining a biological sample from the
subject, determining if microRNA associated with the presence of
oncogenic HPV in the biological sample shows a change in expression
relative to controls, and providing treatment of oncogenic HPV
infection for subjects identified as exhibiting a change in
expression levels of the microRNA. Methods of treating infection by
oncogenic HPV include treatment with antiviral agents, or if
precancerous growth is present, treating the precancerous cells
with cryotherapy, conization, or Loop Electrosurgical Excision
Procedure (LEEP).
Kits
[0072] The present disclosure also provides kits for detecting
oncogenic human papillomavirus in a subject. The kits include one
or more primers and/or probes capable of hybridizing with microRNA
associated with oncogenic HPV, and a package for holding the
primers or probes. A kit generally includes a package with one or
more containers holding the reagents, as one or more separate
compositions or, optionally, as an admixture where the
compatibility of the reagents will allow. The kits may further
include enzymes (e.g., polymerases), buffers, labeling agents,
nucleotides (dNTPs), controls, and any other materials necessary
for carrying out the detection of oncogenic HPV. Kits can also
include a tool for obtaining a sample from a subject, such as a
suitably sized vessel for providing and receiving an oral
sample.
[0073] In an exemplary embodiment, detection kits comprising
polynucleotides attached or immobilized to a solid support. In
another embodiment, detection kits are based on a hybridization
assay. In a further embodiment, detection kits are based on a
reverse hybridization assay.
[0074] The kit for the oncogenic HPV may further comprise any
element, such as reagents used for a microarray method, including,
for example, a reagent used for RNA-labeling reaction, a reagent
used for hybridization, a reagent used for washing, and a reagent
used for extracting RNA from a tissue in addition to the
above-described microarray. The microarray method can be
specifically exemplified by a method which involves measuring the
expression level of a microRNA on DNA Microarray Scanner (from
Agilent Technologies) using Agilent Human miRNA V2 (from Agilent
Technologies) according to the method described in Agilent
Technologies' miRNA Microarray Protocol Version 1.5. The microarray
to which the probe consisting of the polynucleotide or a part
thereof is fixed can be prepared, for example, by synthesizing a
polynucleotide based on the sequence information of the present
microRNA to be measured and fixing it to a commercially available
array.
[0075] In some embodiments, a kit comprises one or more pairs of
primers (a "forward primer" and a "reverse primer") for
amplification of a cDNA reverse transcribed from a target RNA for
carrying out PCR or RT-PCR. Accordingly, in some embodiments, a
first primer comprises a region of at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, or at least 10 contiguous
nucleotides having a sequence that is identical to the sequence of
a region of at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, or at least 10 contiguous nucleotides at the
5'-end of a target RNA. Furthermore, in some embodiments, a second
primer comprises a region of at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, or at least 10 contiguous
nucleotides having a sequence that is complementary to the sequence
of a region of at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, or at least 10 contiguous nucleotides at the
3'-end of a target RNA. In some embodiments, the kit comprises at
least a first set of primers for amplification of a cDNA that is
reverse transcribed from a target RNA capable of specifically
hybridizing to a nucleic acid comprising a sequence identically
present in one of SEQ ID NOs: 1 to 13 and/or a cDNA that is reverse
transcribed from a target RNA.
[0076] In some embodiments, the kit comprises at least two, at
least four, at least 10, or at least 13 sets of primers, each of
which is for amplification of a cDNA that is reverse transcribed
from a different target RNA capable of specifically hybridizing to
a sequence selected from SEQ ID NOs: 1 to 13 and/or a cDNA that is
reverse transcribed from a target In some embodiments, the kit
comprises at least one set of primers that is capable of amplifying
more than one cDNA reverse transcribed from a target RNA in a
sample.
[0077] In some embodiments, probes and/or primers for use in the
compositions described herein comprise deoxyribonucleotides. In
some embodiments, probes and/or primers for use in the compositions
described herein comprise deoxyribonucleotides and one or more
nucleotide analogs, such as LNA analogs or other duplex-stabilizing
nucleotide analogs described above. In some embodiments, probes
and/or primers for use in the compositions described herein
comprise all nucleotide analogs. In some embodiments, the probes
and/or primers comprise one or more duplex-stabilizing nucleotide
analogs, such as LNA analogs, in the region of complementarity.
[0078] In some embodiments, the kits for use in RT-PCR methods
described herein further comprise reagents for use in the reverse
transcription and amplification reactions. In some embodiments, the
kits comprise enzymes such as reverse transcriptase, and a heat
stable DNA polymerase, such as Taq polymerase. In some embodiments,
the kits further comprise deoxyribonucleotide triphosphates (dNTP)
for use in reverse transcription and amplification. In further
embodiments, the kits comprise buffers optimized for specific
hybridization of the probes and primers.
[0079] The kit can also include instructions for using the kit to
carry out a method of detecting oncogenic HPV in a subject.
Instructions included in kits can be affixed to packaging material
or can be included as a package insert. While the instructions are
typically written or printed materials they are not limited to
such. Any medium capable of storing such instructions and
communicating them to an end user is contemplated by this
disclosure. Such media include, but are not limited to, electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. As used herein, the
term "instructions" can include the address of an internet site
that provides the instructions.
[0080] Examples have been included to more clearly describe a
particular embodiment of the invention and its associated cost and
operational advantages. However, there are a wide variety of other
embodiments within the scope of the present invention, which should
not be limited to the particular examples provided herein.
EXAMPLES
Example 1
[0081] In operation, in an exemplary embodiment an in vivo
orthotropic model of aggressive, poorly differentiated tongue
squamous cell carcinoma was established and used to perform miRNA
array profiling to identify microRNAs important in disease
progression. These results identified 29 differentially expressed
miRNAs (18 increased, 11 decreased), two of which were validated by
qPCR: miR-146a (6-fold down-regulation) and miR-452 (7-fold
upregulation). Expression of miR-146a was low in 3 distinct
aggressive oropharyngeal squamous cell carcinoma (OSCC) cell lines
relative to normal oral epithelium. Because these universally
expressed non-coding RNA are stable across samples, it is concluded
that miRNA is relatively stable and protected in paraffin, as shown
in FIG. 1. Therefore, one can detect biologically relevant
differences in miRNA between samples. This was validated by
fluorescent in situ hybridization analysis of formalin-fixed
paraffin-embedded (FFPE) human tissues showing loss of miR-146a.
Restored expression of miR-146a using lentiviral vectors that do
not result in supra-physiological expression levels and have shown
that restoration of miR-146a results in an 8-fold decrease in
invasion in vitro and reduced tumor growth in vivo. Results of
up-regulated are shown in Table 2. Results of down-regulated are
shown in Table 3. The results are graphically represented in FIGS.
2 and 3.
TABLE-US-00002 TABLE 2 "UP" logFC FC P-value miR-320a 1.50 2.83
0.002 miR-222-3p 1.20 2.31 0.004 miR-93-5p 1.24 2.36 0.005
[0082] With regard to miR-320a, the microarray data from 6 OPSCC
cell lines supports upregulation in HPV+ cell lines compared to
HPV-. A proposed role for miR-320a may be in affecting B-catenin,
and may act in a redundant manner with the miR-200 cluster
(significantly up regulated in HPV+ cell lines). With regard to
miR-222, this miR is a family member with miR-221, may regulate
radiosensitivity, and cell growth and invasion, and includes
p27kip1 as a validated target. MiR-93-5p was upregulated in 2
cervical cancer studies, and is a member of the miR-106b cluster,
which likely has considerable biological redundancy with the
miR-17-92 cluster.
TABLE-US-00003 TABLE 3 "DOWN" logFC FC P-value miR-451a -3.7 0.07
0.003 miR-199a-3p// -2.86 0.13 0.005 199b-3p miR-199b-5p -2.85 0.13
0.008 miR-145-5p -2.96 0.20 0.005 miR-143-3p -2.27 0.20 0.002
miR-126-5p -2.14 0.22 0.005 miR-126-3p -1.93 0.26 0.006
[0083] With regard to the downregulated miR, miR-451 is
significantly upregulated in the saliva of patients with esophageal
SCC1. The role of miR-199a is unclear, but it is most likely
involved in modulating metastatic genes, and was shown in one study
to be downregulated in both cervical and HNSCC2. miR-143/145 was
down-regulated in three cervical cancer studies and at least one
HNSCC study, and has been shown to be a tumor suppressor (and
downregulated) in esophageal SCC3. miR-126 likely modulates
Pi3K/Akt/mTOR, and was down-regulated in 3 studies in cervical
cancer
[0084] In an exemplary embodiment, a cohort of samples were
collected, using the following protocol. A new pair of nitrile or
latex gloves was used for each sample collection, Scope.RTM.
mouthwash, Saline solution used as an alternative to Scope for
participants with oral ulcers or those unable to tolerate
mouthwash, and a 2 ounce sterile medicine cup for dispensing
mouthwash. Also needed was a 5 ounce sterile specimen container to
spit the mouthwash sample into and store the sample. A bar-code
label was applied to each specimen container with a de-identified
number code to catalog the sample while maintaining
confidentiality. Finally, a consent form, a cooler with ice for
sample storage, and a timer or stopwatch were used. Participants
aged eighteen and older were eligible for the oral rinse component.
There were no additional exclusion criteria for this exam. Gloves
were put on and mouthwash was poured into a medicine cup, making
sure not to touch the rim of the cup. The medicine cup with the
mouthwash was handed to the participant. When the participant was
ready, she/he was instructed to put mouthwash in mouth and swish
for 15 seconds. After 15 seconds, the participant was instructed to
begin gargling for 15 seconds. When time was up, the specimen
container was opened and handed to the participant. The top was
held lid-down to avoid contamination. The participant spit the
mouthwash into the specimen container. The specimen container was
taken from the participant without touching the rim. The specimen
container was sealed properly to avoid leakage. The bar-code label
was placed on the specimen container. In a preferred embodiment,
the subject has consumed any food or beverage in the hour before
the sample was collected.
[0085] The fluid sample was transferred into a 50 mL conical tube
with corresponding barcode number. Using automatic auto cell
counter, background count and cell number count was obtained from
each sample. 0.5 mL of the oral rinse sample was mixed into 4.5 mL
buffer solution. The tubes were centrifuged at 40,000 rpm for five
minutes. The appropriate balance from the centrifuge was verified.
The supernatant fluid was removed and aliquoted into nine barcoded
cryo vials. Each barcode was scanned into database, matching with
corresponding sample barcode number. Cryo vials were then stored in
liquid nitrogen freezer at -81 degrees. The cell plug was washed
with 20 mL of PBS three times. The cells were centrifuged at 1200
rpm for 2 minutes. The liquid was then discarded and cells were
re-suspended to reach approximately 10,000 cells per mL. The
cytospin centrifuge was set up with Cytopro chamber cups and white
absorption pad. Six slides were labeled with barcode numbers and
slide a, b, c as needed. Each sample has six slides for cytospin.
0.3 mL of sample was placed into small well of cytopro chamber. The
centrifuge was run at 1000 rpm for 5 minutes. At least one aliquot
of the cell pellet from each sample was frozen in small conical
tubes and placed in the liquid nitrogen freezer at -81.degree.
Celsius for later analysis. Slides were removed and discarded
cytopro chambers and absorption pads were discarded, also. Slides
were fixed in 4% PFA (stock was 16%/10 mL, mixed with 30 mL PBS)
for 10 to 20 minutes. Slides were washed with PBS for 2 minutes.
Once they were dried, slides were stored in slide box at
-81.degree. Celsius freezer in Tissue Core.
Example 2
[0086] In an exemplary embodiment, validation of microRNAs occurs
in a three-fold process. First, PCR is conducted on HPV+ compared
to HPV- cases with 24 patients. Next, the model "miR
expressions=HPV+HPV (smokers)+age" was used to determine the
likelihood of miR expression based on the three factors of HPV,
smoking, and age. Third, a bioinformatics analysis was performed
using publically available databases, such as The Cancer Genome
Atlas (TCGA), for example. To test which miRNAs were differentially
expressed based on HPV status (for the FFPE PCR profiling), a
linear modeling framework designed for high-throughput biological
experiments was used. Specifically, using the R/Bionconductor limma
package, moderated t-statistics were applied to each microRNA using
an Empirical Bayes approach, in which the standard errors are
shrunk towards a common value (Smyth G K 2005 & Smyth, G K
2004). The comparison of interest was HPV+vs. HPV-, expressed in
terms of fold change (HPV+/HPV-). The comparison was made by
adjusting for smoking, smoking and HPV interaction, and age to
account for potential confounding effects and expressed as the
equation: miRexp=smoking+HPVstatus+smoking*HPVstatus
(interaction)+age. One such cohort is shown in Tables 4a and
4b.
TABLE-US-00004 TABLE 4a Family Primary or Cell line ID Age Gender
Ethnicity Smoking Alcohol Hx Recurrence SCC003 65 F Caucasian Y Y N
NP SCC072 61 F Caucasian Y Y Y NP SCC090 46 M Caucasian Y Y Y R
SCC200 74 M Caucasian Y Y N NP
TABLE-US-00005 TABLE 4b Path 11q13 TP53 Cell line ID Site Grade
Stage amp. codon HPV+ SCC003 TONS 1 T1NO Y WT -- SCC072 TONS 2
T2N2B Y Mut -- (179) SCC090 BOT 3 T2NO N WT + SCC200 TONS 1 T2N2B N
WT +
Example 3
[0087] In an exemplary embodiment, quantitative reverse
transcriptase polymerase chain reaction (qRT-PCR) is used as a
detection technique. The objective was to validate the loss or gain
of the most differentially expressed microRNAs utilizing (i) in
vitro cell line systems designed to recapitulate salient phenotypic
features of OPSCC and (ii) an expanded human tissue cohort. In an
alternate embodiment, an expanded human saliva sample cohort could
be used. Total RNA was extracted from various cell lines using the
miRCURY.TM. RNA Isolation Kit--Cell and Plant (Product number
300110, Exiqon A/S, Denmark). All cells were taken at 50%
confluence on 10 cm plates as recommended by the manufacturer.
[0088] RNA from 10 cell lines (SCC200, 090, 036, 152, HTE clone
21505, SCC003, 072, 089, 103, and HTE clone D) in technical
replicates of three, was extracted utilizing miRCURY RNA Isolation
kit (Exiqon) according to the manufacturer's instructions for a
total of 30 RNA samples. This kit utilized spin column
chromatography using a resin to separate total RNA, including mRNA,
rRNA, and other small RNAs, from other cell components without the
use of phenol, trizol, or chloroform. Each of the 30 samples was
polyadenylated and reverse transcribed into complementary DNA in a
single reaction with the Universal cDNA Synthesis kit II (Exiqon).
A synthetic RNA spike-in, UniSp6, was added to each sample as a
means to monitor RT efficiency and reproducibility in the final
qPCR experiments.
[0089] Pick-&-Mix microRNA PCR Panel (Exiqon, product #203801
and 203802) consist of 96-well PCR plates containing custom
selections of dried down microRNA LNA.TM. PCR primer sets for one
10 .mu.L real-time PCR reaction per well, ready-to-use. The LNA.TM.
primer sets are designed for optimal performance with the Universal
cDNA Synthesis Kit II and the ExiLENT SYBR.RTM. Green master mix
kit. Primer sets for 18 microRNAs of interest were selected based
on microarray data and the FFPE PCR profile. Also included, were 4
candidate reference genes and 2 positive control primer sets, for a
total of 8 plates. Each of the 8 plates thus contained wells to
assay 24 PCR reactions for 4 samples. The 30 samples were divided
into groups of 4 different biological replicates such that cDNA
from a single cell line was dispersed between 3 plates. Two
negative control samples were included on plate 8, one no template
control (NTC) and one sample that was run without reverse
transcription (No Enzyme control).
[0090] Prior to dispensing samples across the plates, cDNA from the
RT reactions were diluted 100.times. with nuclease free water. cDNA
and 2.times.PCR master mix (Exiqon) were combined 1:1 and 10 mcls
added to each well, corresponding to 0.05 ng total RNA per PCR
reaction. The plate was then sealed as recommended by the PCR
instrument manufacturer and spun in a plate centrifuge for 1
minute.
[0091] Real-time PCR Amplification and melting curve analysis was
performed on an ABI Step One Plus in a standard (2 hr) run
according to the following cycles: (1) polymerase
activation/denaturation at 95.degree. C. for 10 minutes, (2)
amplification for 40 cycles at 95.degree. C. for 10 seconds
followed by 60.degree. C. for 1 minute at a ramp-rate of
1.6.degree. C./s, and (3) melting curve analysis as specified by
the StepOne Plus system. The ABI system was set to manual baseline
and threshold as opposed to auto Ct settings as per the
recommendation of Exiqon. Raw Ct values, amplification, plate
setup, melt region temperature, melt region normalized, and melt
region derivative data were exported as .txt and .xls files for
data analysis.
[0092] The results showed an upregulation of SEQ IDs 1-9, and a
down-regulation of SQ IDs 10-13 in samples of subjects with
oncogenic HPV. FIG. 5 demonstrates the results of miR-145. The
miR-145 sequence in E1 showed a significant reduction (80%) in
luciferase activity with increasing levels of miR-145 (B), whereas
the E2 region showed a slight reduction in luciferase activity (C).
The data are from three independent experiments, and standard
errors are shown.
[0093] The results of the HPV+/HPV- comparison were first sorted
according to adjusted p-value, which is the Benjamini-Hochberg
false discovery rate (fdr) adjustment that accounts for the large
number of tests performed, as shown in Table 5. For this, an
fdr=0.2 was chosen since validation studies were planned, although
0.3 or 0.4 would have been considered acceptable given the planned
validation efforts. Moderated T tests were performed to produce
p-values for each microRNA comparing HPV+ to HPV-.
TABLE-US-00006 TABLE 5 Adj. P. Val ID logFC P. Value (aka FDR)
PC_positive miR-320a 1.501044432 0.002024594 0.20380063 2.830475493
miR-143-3p -2.277057625 0.002502535 0.20380063 0.206318111 miR-451a
-3.705247757 0.003195508 0.20380063 0.076667144 miR-222-3p
1.205682839 0.004577297 0.20380063 2.306464103 miR-93-5p
1.237836139 0.005405395 0.20380063 2.358445298 miR-199a-3p//
-2.866705587 0.005316526 0.20380063 0.137099424 miR-199b-3p
miR-126-5p -2.142609506 0.005480287 0.20380063 0.226469786
miR-145-5p -2.296799107 0.005907265 0.20380063 0.203514133
miR-126-3p -1.939807196 0.006935735 0.212695871 0.260651272
miR-199b-5p -2.850689997 0.008802817 0.242957761 0.138629866
Example 4
[0094] In an exemplary embodiment, a microfluidics device is used
as a detection technique. 15 mL of a saliva sample, collected from
a human subject, was run through a filter (from Milipore, Code
VVLP, size 100 nm), separating all cells. The filtered cells were
then collected. 50 .mu.L of lysis buffer, (BP-200 from Boston
Bioproducts) was added to the filtered cells, and the cells are
lysed. Then, 50 .mu.L of glycerol (from SigmaAldrich) was added to
the solution. Following the addition of glycerol, 100 .mu.L of this
lysate was pipetted onto the microfluidics chip. All electrodes and
fluidic connections were placed at their positions, and reservoirs
on the chip were filled with buffer. An electrochemical baseline of
the sensor is measured. Next, an extraction of the target microRNAs
from the lysate took place by applying an electrical field. The
extracted target microRNAs were concentrated at the sensor by
applying convective flow and an electrical field on the system. The
target molecules hybridized on the short RNA probes on the sensor.
All non-target molecules were washed off the sensor. An
electrochemical measurement of the sense was conducted to determine
the change compared to the baseline. The size of the change
indicated the concentration of target molecules in the sample.
[0095] The results showed a change in the output electrochemical
curve, shifting the baseline curve to the right, indicating the
presence of the target microRNAs.
Example 5
[0096] In an exemplary embodiment, in situ hybridization (ISH) is
used as a detection technique. For the tissue-based in situ
hybridization studies, the aim was to assess the expression of
microRNAs that were statistically significant between HPV+vs HPV-
oropharyngeal squamous cell carcinoma (OSCC) and expressed at
relatively high levels according to miRNAseq data utilizing (i) in
vitro cell line systems designed to recapitulate salient phenotypic
features of OPSCC and (ii) an expanded human tissue cohort. In an
alternate embodiment, an expanded human saliva sample cohort could
be used.
[0097] Head and neck squamous cell carcinoma (HNSCC) tissue
microarrays (TMA) were provided by Dr Jim Lewis (Department of
Pathology & Immunology, Washington University School of
Medicine, St Louis, Mo.). The TMA included 357 cases of HNSCC with
two tumor tissue cores per case, thus compromised 17 formalin-fixed
paraffin embedded tissue blocks (FFPE). Each block was sectioned
onto four separate slides (4 .mu.m thick), for a total of 68 slides
and sent to The Center for RNA interference and Non-Coding RNAs at
MD Anderson Cancer Center (MDACC, Houston, Tex.) for ISH
hybridization and staining. The probes for in situ hybridization
(ISH) were ordered from Exiqon (Denmark) and shipped directly to
MDACC. The probes were selected based on the results of FFPE, TCGA,
and cell line profiling based on their high relative expression
levels and statistically significant differences between HPV+ and
HPV- tumor tissues. Double digoxigenin (DIG) labeled (5' and 3')
miRCURY LNA.TM. probes are optimized for detection of microRNAs in
FFPE tissue sections. For visualization, the digoxigenins are
detected with a polyclonal anti-DIG antibody and alkaline
phosphatase-conjugated secondary antibody using
5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium
(BCIP/NBT).
[0098] In these experiments, the full-length mature microRNA
sequences were used for three specific locked nucleic acid (LNA)
probes:
TABLE-US-00007 SEQ ID No.: 27 TCATACAGCTAGATAACCAAAGA;, SEQ ID No.:
28 ATCTGCACTGTCAGCACTTTA;, SEQ ID No.: 29
GAACAGGTAGTCTGAACACTGGG,
[0099] Optimization of binding conditions was performed by MDACC,
but briefly the tissue is first digested briefly with Protease K.
Next, the probes are hybridized to their targets. Followed by
AP-conjugated antibodies and BCIP/NBT development to produce purple
signal. All tumor tissues stained for the three microRNAs were
counterstained with nuclear fast red. One entire TMA was stained
with LNA U6 snRNA probes as positive controls without counterstain.
Each TMA contained normal reference tissues that served as negative
controls (liver, thyroid, and small bowel).
[0100] The stained slides were shipped, read, and scored in a
systematic fashion according to the following 3 parameters: (1)
proportion of tumors cells with identifiable ISH signal (0<10%,
1=10-25%, 2=25-50%, 3=50-75%, 4=>75%); (2) strength of ISH
signal (weak, weak/moderate, moderate, strong); (3) staining
pattern (punctate, diffuse, or punctate+diffuse). All images were
assessed in a blinded fashion as to HPV status, H&E morphology,
and all other clinicopathological parameters.
[0101] The staining results were tabulated on a .csv file and then
compared to prior data concerning HPV status. The subsequent
analysis was performed. Logistic regression analysis was performed
separately for each binary outcome (HPV status measured by p16 and
ISH) with the following for predictors: staining proportion,
strength of ISH signal, and staining pattern. Model selection
criteria were used to guide the selection of the final variables
included in the model. Pseudo-R.sup.2 and internal cross validation
were reported to indicate the performance of the model.
[0102] Results showed hybridization of the probes to microRNA in
the samples of subjects with oncogenic HPV. Those samples not
infected with oncogenic HPV did not hybridize to the probes.
Example 6
Identification of Human Papillomavirus-Associated Oncogenic
microRNA Panel in Human Oropharyngeal Squamous Cell Carcinoma
Validated by Bioinformatics Analysis of the Cancer Genome Atlas
[0103] Expression profiling of cancers and functional studies
performed in cancer cell lines and murine models have revealed
provocative patterns implicating miRNA-mRNA dysregulation as
important in tumor development and progression. Somewhat
independent of miRNA biology, miRNA profiles offer diagnostic and
prognostic utility in cancer and other diseases that may guide
treatment. Schetter et al., JAMA., 299(4): 425-36 (2008); Schultz
et al., JAMA., 311(4):392-404 (2014). To this end, the objective of
this study was to profile HPV+ versus HPVOPSCC to provide a more
detailed understanding of pathological molecular events and to
identify biomarkers that may have applicability for early
diagnosis, improved staging, and prognostic stratification. We
identify an "oncogenic miRNA panel" that represents the host
response to an oncogenic HPV infection and validate this panel in
additional clinical cohorts using publicly available sequencing and
clinical data from The Cancer Genome Atlas (TCGA) and miRNA-in situ
hybridization (miRNA-ISH) analysis of arrayed human OPSCC tissues.
This molecular signature may have utility to differentiate
oropharyngeal tumors with different prognoses and thus distinct
management strategies and facilitate mechanistic elucidation of
molecular factors that contribute to OPSCC development, progression
and response to therapy.
[0104] Materials and Methods
[0105] Patient Samples for miRNA Profiling. Tissues for the initial
study cohort were obtained from University of Missouri surgical
pathology archives (2006-2011) with Institutional Review Board
approval and represent histologically confirmed tonsillar or base
of tongue squamous cell carcinoma (OPSCC). Tissue for study was
identified by staining for p16 according to the manufacturer's
instructions (CINtec Histology Kit, MTM laboratories; E6H4 clone;
Ventana Medical Systems, Roche) and evaluated using a binary rating
system, with positive representing extensive (>50%) tumor-cell
specific cytoplasmic and nuclear staining. Negative staining
represented sparse or absent tumor-specific staining. `Focal
staining patterns`, in the presence of mostly negative staining was
interpreted as negative. All cases included for laser capture
microdissection represented unambiguous staining patterns.
[0106] Laser Capture Microdissection.
[0107] Using p16 staining we identified cases from surgical
excisions that were strongly positive (>90% immunoreactivity for
p16 with minimal criteria=>70%) or completely negative. Of the
109 cases stained, 53 had sufficient primary tumor tissue available
for laser capture, performed on the ArcturusXT system (Invitrogen).
For each case, a minimum of 8000 cancer cells were dissected and
caps stored at -80 C. RNA purification utilized the miRNeasy FFPE
kit (Qiagen). All samples were assessed with a Nanodrop
spectrophotometer and Agilent Bioanalyzer 2100. Additionally, a
limited number of samples were run on a miRNA QC PCR array as a
means of positive control to assess stability of the small RNA
fraction (Qiagen).
[0108] Real Time PCR Based miRNA Profiling.
[0109] Qiagen's miRnome global qPCR array, which includes assays
for 1008 individual microRNA species, was utilized for profiling.
As described below, each array consisted of three 384-well plates,
such that each patient's sample was run on three separate plates.
The 1008 sequences profiled represent all annotated miRNA sequences
in the human miRNA genome (miRBase release 16). cDNA was
synthesized with the miScript II RT kit and qPCR was performed
using the Roche LightCycler480. Each assay was begun with hot start
activation and included 45 cycles of amplification with melt curve
analysis step for determining specificity of each probe (by
calculating Tm). Absolute quantitation was used to generate
threshold values (Cp) using the second derivative maximum algorithm
unique to the LC480 system (auto baseline). Statistical analysis
was performed using the geNorm algorithm. To improve the threshold
of detection, pre-amplification was performed on cDNA synthesized
with the miScript II RT kit using the miScript PreAMP PCR kit and
miScript PreAMP Primer mix (Qiagen). Amplified cDNA was added to
the miScript SYBR mix, water and MiScript universal primer and
dispensed to miRNome plates. A test miRNome array was first
utilized to determine if the dilution for real time PCR resulted in
high percentage call rates. Pre-amplified samples including
p16+(n=15) and p16- (n=9) samples were loaded on the miRnome plates
(15 ng cDNA/sample). Subsequent analysis was performed as described
above using Ct values.ltoreq.30 as a cutoff for inclusion.
[0110] PPPE-Based miRNA Cohort Bioinformatics.
[0111] Raw data from the RT-PCR arrays were subjected to extensive
quality control analyses based on specialized internal controls on
the arrays including positive PCR controls, which test the
efficiency of the polymerase chain reaction itself, and reverse
transcription controls to detect any impurities that inhibited the
RT phase of the procedure. We also calculated mean, standard
deviation, and coefficient of variation and compared them to values
published on FFPE cancer samples for these arrays. Philippidou et
al, Cancer Res., 70(10):4163-73 (2010). Any sample that failed to
fall within the acceptable range of metrics as defined by Qiagen
was excluded from the analysis; one of our 24 samples failed this
step. Next, miRNAs with Ct values of >30 (or 0) for
pre-amplified samples were considered `not reliably detected` and
excluded from analysis by replacing that Ct with `NA` to indicate
missing. Determining reference genes for normalization was carried
out on a plate-by-plate basis according to the geNorm algorithm,
utilizing the R/Bioconductor package SLqPCR. Peltier, H J, Latham,
G J, RNA, 14(5):844-52 (2008). To test which miRNAs were
differentially expressed based on HPV status, we used the
R/Bioconductor limma package. To focus on widely expressed miRNAs
and to increase power, non-specific filtering of miRNAs was carried
out as follows: only those miRNAs which were detected in >90% of
samples were carried forward for subsequent analysis. Moderated
t-statistics were applied to each miRNA using an Empirical Bayes
approach, in which the standard errors are shrunk towards a common
value. The comparison of interest was HPV+ versus HPV-, expressed
in terms of fold change (HPV+/HPV-). The comparison was made after
adjusting for smoking, smoking and HPV interaction, and age to
account for potential confounding effects. In the event of a
significant interaction between smoking and HPV for a specific
miRNA, the interpretation of the effect of either smoking or HPV in
isolation should be made with caution.
[0112] Validation of Oncogenic miRNA Panel Using Patient Cohorts
from the Cancer Genome Atlas (TCGA).
[0113] Patient consent/enrollment and utilization of data were
conducted in accordance with TCGA Human Subjects Protection and
Data Access Policies. Clinical data were first downloaded for HNSCC
patients (458 cases), HPV ISH or HPV p16 testing was assessed, and
then patients with definitive status (positive or negative) were
selected resulting in 66 cases. Of these, 11 cases were positive
and 11 were definitively HPV- according to p16 and/or HPV- ISH.
Annotated RNA-Seq data were then downloaded from the TCGA Data
Portal in September and October 2013. Because the comparison of
interest was HPV+ to HPV- cancer, individual normalized expression
values for a particular miRNA were compared between groups of HPV+
patients (n=11) and HPV- patients (n=11) by using student's t-test
and the results sorted according to the level of significance. For
miRNAseq, the relative abundance of a particular microRNA is
represented by the absolute number of sequence reads.
[0114] Validation by miRNA-In Situ Hybridization (miRNA-ISH) in
HNSCC Tissue Microarrays.
[0115] HNSCC tissue microarrays (TMA) were assembled from cases
available in the Department of Pathology & Immunology,
Washington University School of Medicine, using tissues obtained
with approval of the Human Research Protection Office. The TMA
included 357 cases of HNSCC with two tumor tissue cores per case
Immunohistochemistry was performed for p16 on a full FFPE section
on a Ventana Benchmark automated immunostainer (Ventana Medical
Systems, Inc., Tucson Ariz.) according to standard protocols with a
known p16-expressing SCC case and normal tonsil as positive and
negative controls, respectively. Antigen retrieval utilized the
Ventana CC1, EDTA-Tris, pH 8.0 solution. Staining was read by one
study pathologist (JSL), and all positive cases demonstrated both
nuclear and cytoplasmic staining. Staining was graded in a quartile
manner as follows: 0=no staining, 1+=1 to 25% staining; 2+=26 to
50%; 3+=51 to 75%, and 4+=76 to 100%. Cases were then classified as
positive (any convincing expression) versus negative, and then
separately as strong positive staining (3 or 4+) versus negative or
weak staining (0, 1+, or 2+). More than 90% of cases were either
strongly and diffusely positive or completely negative. The TMAs
were then processed for RNA-ISH for HPV or miRNA-ISH for miR-9. In
situ hybridization for high risk HPV E6/E7 RNA was performed using
the RNAscope.TM. HPV kit (Advanced Cell Diagnostics, Inc., Hayward,
Calif.) according to the manufacturer's instructions and classified
by the study pathologist (JSL) as either positive or negative.
Positive cases had granular cytoplasmic and/or nuclear brown
staining that was above the signal on the negative control slide.
In situ hybridization for miR-9 was performed at the Center for RNA
Interference and Non-Coding RNAs at MD Anderson Cancer Center
(MDACC, Houston, Tex.). Double digoxigenin (DIG) labeled (5' and
3') miRCURY LNA.TM. probes were obtained from Exiqon (Denmark). For
visualization, the digoxigenins are detected with a polyclonal
anti-DIG antibody and alkaline phosphatase conjugated secondary
antibody using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue
tetrazolium (BCIP/NBT). In these experiments, the full-length
mature microRNA sequence for miR-9 was used for specific LNA
probes: TCATACAGCTAGATAACCAAAGA (SEQ ID NO: 27). All tumor tissues
stained for miR-9 were counterstained with nuclear fast red. One
entire TMA was stained with LNA U6 snRNA probe as positive controls
without counterstain. Each TMA contained normal reference tissues
that served for orientation and as negative controls (liver,
thyroid, or small bowel). All resulting slides were assessed (by
DLM and/or JSL) in a blinded fashion as to HPV status, H&E
morphology, and all other clinical-pathologic parameters. The
stained slides were read and scored in a systematic fashion
according to the following 3 parameters: (1) proportion of tumors
cells with identifiable ISH signal (0<10%, 1=10-25%, 2=25-50%,
3=50-75%, 4=>75%); (2) strength of ISH signal (weak,
weak/moderate, moderate, strong); (3) staining pattern (punctate,
diffuse, or punctate+diffuse). The staining results were tabulated
then unblinded and combined with data regarding HPV status and
additional clinical data. The subsequent logistic regression
analysis was performed separately for each binary outcome (HPV
status measured by p16 and ISH) with the following for predictors:
staining proportion, strength of ISH signal, and staining pattern.
Model selection criteria were used to guide the selection of the
final variables included in the model. Internal cross-validation
was reported to indicate the performance of the model.
[0116] Results
[0117] Characterization of Oropharyngeal Tissues.
[0118] As national and international rates of HPV driven OPSCC have
been called epidemic in scale, we therefore asked whether patients
previously diagnosed with OPSCC at the University of Missouri
reflect concordant epidemiological proportions. Of the cases
(n=109) stained for p16 expression, 58% were positive (FIG. 17A).
Multi-institutional US studies estimate 65-70% of OPSCC to be
caused by HPV. Chaturvedi et al., J Clin Oncol, 29: 4294-301
(2011). The average ages in the cohorts under study were 56.49 and
61.00 years of age for p16+ and p16- respectively (FIG. 17B).
HPV/OPSCC has a male predominance and no consistent association
with smoking. Disease staging also has been associated with
differences between HPV+ and HPV- OPSCC, with HPV+ disease more
commonly associated with stage IV disease and frequent lymph node
metastases at initial presentation. Overall, we interpret these
clinical data as positive signal that our p16+ and p16- cohorts
reflect true disease trends.
[0119] HPV+ and HPV- Tumors have Distinct MiRNA Profiles.
[0120] To identify a distinct miRNA signature that can
differentiate HPV+ from HPV- OPSCC, we performed PCR-based miRNA
profiling using a minimum of ten 10 .mu.m sections from each of 24
cases. Following preamplification, improved signal detection is
evident. One case was excluded based on quality control measures.
Prior to non-specific filtering there were 511 miRNAs; afterward
276 remained and were used for modeling. Results from our linear
model showed that three individual miRNA sequences were
significantly up-regulated in HPV+ patients: miR-320a, miR-222-3p,
and miR-93-5p. The most statistically significant downregulated
miRNAs included 6 sequences, representing 4 unique mature miRNAs:
miR-199a-3p//-199b-3p, miR-143, 145, and mir-126a (FIG. 18A,B and
Table 6). The top 10 miRNAs that were most impacted by age or
smoking status do not show fold changes of the magnitude we found
associated with HPV status. Three miRNAs that showed a significant
HPV effect also had a significant Smoking.times.HPV effect
(miR-320a, miR-126, and miR-143; results not shown). The full
expression matrix (511 miRNAs, 23 samples) was log.sub.2
transformed and clustered using unsupervised hierarchical
clustering based on average agglomeration with |1-rho| as the
distance measure. The results are presented as a heat map, with
dendrograms indicating the clustering of patient samples (columns)
and miRNAs (rows) (FIG. 18A). Additional unsupervised hierarchical
clustering was performed using a list of 43 miRNAs implicated by
our own dataset as well as those from the literature (FIG. 18C).
These analyses resulted in data that strongly supports the
hypothesis that HPV+ oropharyngeal tumors display distinct miRNA
profiles and that groups of miRNAs can be associated with an
oncogenic HPV infection.
TABLE-US-00008 TABLE 6 Oncogenic miRNA profile from FFPE HPV+ vs
HPV- OPSCC cases. Table depicts fold change and log.sub.2FC for the
9 most statistically significant miRNA sequences, representing 7
distinct miRNAs. Fold Change logFC p-value hsa-miR-320a 2.83 1.50
2.02E-03 hsa-miR-143-3p 0.21 -2.28 2.50E-03 hsa-miR-222-3p 2.31
1.21 4.58E-03 hsa-miR-93-5p 2.36 1.24 5.41E-03 hsa-miR-199a-3p//
0.14 -2.87 5.32E-03 hsa-miR-199b-3p hsa-miR-126-5p 0.23 -2.14
5.48E-03 hsa-miR-145-5p 0.20 -2.30 5.91E-03 hsa-miR-126-3p 0.26
-1.94 6.94E-03 hsa-miR-199b-5p 0.14 -2.85 8.80E-03
[0121] Correlation of miRNA Expression with HPV Expression from
TCGA Database Validates PCR-Based HPV- Associated miRNA
Profile.
[0122] To further assess changes in the miRNA expression profile
associated with HPV+OPSCC, we utilized publically available
clinical and miRNA-seq data from The Cancer Genome Atlas (TCGA) and
identified 11 primary OPSCC cases (tonsil or BOT) positive for
either p16 IHC or HPV ISH that were paired with 11 HPVcases, which
we designated as TCGA Cohort 1. In TCGA Cohort 1, we found 84
miRNAs to be significant (p<0.05 and mean RPKM>10 in both
groups). Using the average RPKM for each group as general
estimations of expression level, fold-changes were calculated (+/-)
and cutoffs of log 2 fold changes of +/-1.0 were used, yielding a
list of 36 miRNAs.
[0123] The results from TCGA Cohort 1 were compared to the panel of
miRNAs identified from our PCR profiling of PIPE tissue. The fold
changes of these 7 miRNAs between HPV+ and HPVpatients are
concordant between the two datasets based on both Spearman's rank
correlation (rho=0.85, p=0.02) and Pearson's correlation (r=0.83,
p=0.02; 95% CI, 0.21-0.97) (FIG. 19A). Amongst the most
statistically significant miRNAs from TCGA Cohort 1, 16 of which
are shown in Table 7, miR-199-1, miR-106b, and miR-9 were highly
correlated to patients who are HPV+ irrespective of other
covariates such as age or smoking status (FIGS. 19B, C, D).
Interestingly, miR-9 was upregulated 1.84 fold (p=0.14) in our FFPE
qPCR cohort and it has been identified by two independent published
studies as an HPV- associated miRNA in OPSCC. Therefore, the
upregulation of both miR-9-1 and miR-9-2 in TCGA Cohort 1 miRNAseq
data served as strong validation that this is an HPV-associated
miRNA in OPSCC. Unsupervised clustering performed on the full
miRNAseq data indicated that, in this dataset, HPV+ disease is
associated with distinct miRNA profiles (FIG. 19E). Notably, a
group of miRNAs that are highly expressed in both HPV+ and HPV-
disease, including miR-21, miR-203, and miR-22 clustered at the
bottom of this heat map, implicating these miRNAs as potentially
important in squamous differentiation of HNSCC regardless of HPV
status.
TABLE-US-00009 TABLE 7 TCGA Cohort 1 and Cohort 2 analysis. Table
depicts fold change and log.sub.2FC for the most statistically
significant miRNA sequences from each cohort. RPKM = reads per
kilobase of transcript per million reads mapped. Data Fold Source
microRNA HPV+ HPV- Change logFC p-value TCGA hsa-mir- 1225 542 2.26
1.18 1.7E-05 COHORT 1 106b hsa-mir- 54501 20645 2.64 1.40 4.8E-05
148a hsa-mir-625 306 111 2.76 1.46 8.8E-05 hsa-mir-335 113 41 2.71
1.44 5.4E-04 hsa-mir-9-1 5078 1054 4.81 2.27 5.4E-04 hsa-mir-9-2
5091 1055 4.82 2.27 5.8E-04 hsa-mir-214 13 46 0.30 -1.75 8.9E-04
hsa-mir-337 10 36 0.29 -1.79 1.3E-03 hsa-mir- 24 7 3.49 1.80
1.7E-03 378c hsa-mir-29c 4456 1204 3.70 1.89 2.5E-03 hsa-mir-598 38
14 2.68 1.42 3.3E-03 hsa-mir-107 133 52 2.55 1.35 5.2E-03
hsa-mir-150 2796 746 3.75 1.91 5.6E-03 hsa-mir- 55 18 3.05 1.61
6.6E-03 106a hsa-mir-378 2154 742 2.90 1.54 6.7E-03 hsa-mir-20b 152
9 16.61 4.05 7.4E-03 TCGA hsa-mir-20b 47 7 6.79 2.76 2.6E-04 COHORT
2 hsa-mir-9-2 5287 325 16.26 4.02 5.4E-04 hsa-mir-9-1 5282 326
16.18 4.02 5.4E-04 hsa-mir- 990 587 1.69 0.75 5.4E-04 106b
hsa-mir-574 44 69 0.63 -0.66 6.8E-04 hsa-mir- 101 256 0.40 -1.34
9.0E-04 193b hsa-mir-363 30 7 4.38 2.13 9.3E-04 hsa-mir-16-2 23 11
1.99 0.99 1.7E-03 hsa-mir-15b 521 269 1.93 0.95 1.7E-03 hsa-mir-25
11787 7377 1.60 0.68 1.8E-03
[0124] Further Validation Studies Using TCGA and miRNA-ISH.
[0125] Based on a recent analysis of mRNAseq data from TCGA across
3,775 malignancies for the presence of viral sequences, a second
expanded cohort of patients from TCGA with HPV-associated HNSCC was
identified comprised of 20 cases that lacked available clinical
data regarding p16 or HPV ISH, and were therefore not identified in
our original analyses. Khoury et al., J Virol., 87(16): 8916-26
(2013). However, these 20 cases were shown to express various viral
transcripts (18 cases expressed transcripts from HPV16 and 2 from
HPV33) and have HPV DNA integrated into the host genome, were thus
treated as HPV+, and were compared to an additional set of 29 HPV-
cases (identified based on available p16 and/or HPV ISH data),
herein designated TCGA Cohort 2. After extracting miRNAs that were
expressed at suitable levels in both cohorts (>10 mean RPKM), a
list of 43 miRNAs was generated, the top 10 of which are shown in
Table 7. The fold changes of our 7 miRNA panel from FFPE studies
show concordance between the two datasets based on Spearmans rank
correlation (rho=0.75, p=0.06) and Pearson's correlation (r=0.78,
p=0.03) (FIG. 20A). MiR-9 and miR-106b were again amongst the most
statistically significant miRNAs. Both miR-9-1 and miR-9-2 are
expressed at reads RPKM of .about.5000 in patients whose tumors
express HPV transcripts. This is in stark contrast to patients
whose tumors are negative for p16 IHC and/or HPV ISH, where on
average miR-9 levels are .about.16-fold lower. The tissue level of
miR-9 expression was also assessed via in situ hybridization on
HNSCC tissue microarrays containing 357 cases including 270 OPSCC
cases with known p16 status and 226 cases with known HPV RNA ISH
results. Representative positive and negative punctate and diffuse
staining is shown in FIG. 21A-H. The odds that high-tumor miR-9
expression occurs in the setting of p16+ disease are more than 3
times greater (OR=3.38, p<0.001; 95% CI 1.84-6.26) than the odds
of low-tumor miR-9 expression; similarly, the odds of diffuse miR-9
ISH are nearly four times greater (OR=3.87, p<0.001; 95% CI
2.10-7.20) than the odds of non-diffuse miR-9 ISH (FIG. 21I).
Interestingly, when the odds ratio calculations are based upon HPV
mRNA in-situ hybridization, the ORs increase (OR=4.41, p<0.001,
95% CI 2.30-8.51) and (OR=5.76, p<0.001, 95% CI 2.99-11.36) for
both miR-9 positivity and a diffuse cytoplasmic/nuclear pattern for
miR-9 ISH signal (FIG. 21J). As has been shown in our own analyses
of FFPE samples and analysis of TCGA Cohort 1 (p16/ISH confirmed
cases), HPV-associated tumors are characterized by upregulation of
miRNAs belonging to the miR-106b.about.25 cluster (miR-106b,
miR-93, miR-25). Our validation studies using TCGA Cohort 2
strongly support upregulation of members of this cluster in
association to HPV status. Further, the expression levels of the
closely related miR-106a.about.363 cluster (miR-20b and miR-363)
are tightly correlated to HPV status, although the expression
levels of these miRNAs are low, making it difficult to interpret
the potential biological significance (Table 7). It should be noted
that others have reported upregulation of miR-20b as well as
miR-363 based on qPCR profiling and microarray analyses of FFPE
samples and cell lines, respectively. Wald et al., Head Neck,
33(4):504-12 (2011); Hui et al., Clin Cancer Res, 19: 2154-2162
(2013).
[0126] Discussion
[0127] HPV- Associated miRNA Profiling. The incidence rates for
HPV+OPSCC have increased dramatically from 1988-2004, from 0.8 to
2.6 per 100,000, an increase of 225%. In striking contrast, HPV-
negative cancers have declined 50%. These trends are also apparent
internationally. Thus, it is of great interest to identify specific
upregulated miRNAs characteristic of an oncogenic HPV infection of
the head and neck, as these miRNAs may represent novel
diagnostic/prognostic biomarkers and may provide a more detailed
understanding of the molecular pathogenesis of the disease. We have
provided miRNA profiles of three independent cohorts of patients
(n=94) with SCC of the aerodigstive tract performed with PCR arrays
and next generation deep sequencing. Comparative analysis of these
cohorts strongly supports an HPV-associated upregulation of miR-9
and members of the miR-106b.about.25 cluster and downregulation of
miR-199-1.
[0128] The fundamental mechanism associated with HPV+ disease is a
disruption of cellular differentiation induced by the virus,
wherein the cell acquires resistance to growth inhibition, immune
evasion, subversion of apoptosis, genomic instability, and
ultimately dysregulated proliferation such that viral DNA can
replicate in synchrony with chromosomal DNA. Thus, HPV may causally
modulate miRNAs that then act as central nodes in affecting
numerous genes important in progression and metastatic spread,
resulting in divergent gene regulation. In contrast to cervical
cancer, viral integration is not necessary for initiation of
oncogenesis in OPSCC and episomal HPV DNA appears to be a frequent
occurrence in tonsillar carcinomas. Syrjanen S., J Clin Pathol.,
57(5):449-55 (2004). However, similar to cervical cancer,
transcriptionally-active HPV is linked to the differentiation state
of the host cell and in some circumstances leads to aberrant cell
proliferation and genomic instability that is due in part to
mitotic spindle defects that result in cytogenetic abnormalities.
Duensing et al., Environ Mol Mutagen., 50(8): 741-7 (2009).
Alternatively, miRNAs could be suppressed due to aberrant DNA
methylation or chromosomal disruption. Johannsen E, Lambert P F.,
Virology, 445(1-2):205-12 (2013). As an individual miRNA can affect
hundreds to thousands of genes, many of these often lie in the same
biological pathway. Lal et al., PLoS Genetics 7(11): e1002363
(2011). Thus, specific cellular pathways important for immune
surveillance, mitogenic signaling, or metabolism, that are integral
for HPV infection, could also lead to typified miRNA
expression.
[0129] Several studies have examined miRNA expression in the
context of HPV and head and neck cancer, with heretofore a lack of
consensus between reports. Lajer et al., Br J Cancer, 104(5):
830-40 (2011); Gao et al., Cancer, 119: 72-80 (2013). This may be
due, in part, to the biological redundancy in miRNA function, the
inherent genomic instability characteristic of HPV+ tumors, and the
use of differing profiling methodologies and sample sizes. It is
interesting to note that miR-20b shares significant sequence
homology with miR-106b and miR-93, these miRNAs share the same seed
sequence, and are closely related to miR-363. It has been reported
that members of the miR-106b.about.25 cluster are upregulated
independent of HPV status relative to normal tonsillar epithelium
(n=88 vs n=7). This is in contrast to the current study showing
upregulation of members of the miR-106b.about.25 cluster in
HPV+OPSCC relative to HPV- as determined in the PIPE PCR-based
miRNA profile, TCGA Cohort 1, and TCGA Cohort 2. Recent in vitro
experiments that sought to identify miRNAs altered as a function of
HPV transfection utilized deep sequencing on organotypic raft
cultures derived from normal or HPV- 31-transfected human foreskin
keratinocytes isolated from the same donor, demonstrating 2-3 fold
upregulation of miR-106b and miR-25 in the HPV- transfected
cultures relative to normal. Gunasekharan V, Laimins L A., J Virol.
87(10):6037-43 (2013). In the context of the current human tumor
data, this study strongly supports that these miRNAs are altered as
a function of HPV oncoproteins.
[0130] The TCGA deep sequencing data sets analyzed here add another
dimension to available data on the expression of miRNAs shown to be
statistically significant between HPV+ and HPV- disease, as
normalized read counts provide information on the relative
abundance of a specific miRNA. Thus, it is interesting to note that
miR-363, which has been shown to be upregulated via microarray
analysis in HPV+ disease by two independent studies, shows
relatively low expression overall (<50 RPM in HPV+ and HPV-
cohorts), yet the fold change for this miRNA is quite high. Similar
in this regard is miR-20b, part of the same transcriptional unit
and also previously reported as upregulated in HPV+ disease. In the
current study, we observed upregulation of miR-20b (16- and 7-fold
in TCGA Cohorts 1 and 2, respectively); however, it is also
expressed at relatively low levels. In contrast, miR-106b and
members of the miR-106b.about.25 cluster are expressed at levels
.about.30-fold higher. As the biological significance of these fold
differences are unknown, the relative importance of the two
clusters in HPV+OPSCC should be addressed experimentally, as it is
unclear whether expression of these miRNAs at very low levels may
nevertheless regulate important biological functions.
[0131] Upregulated miRNAs.
[0132] "MiRNA families" are determined based on common seed
sequence and are predicted to target overlapping sets of genes.
miR-106b.about.25 and miR-106a.about.363 are genomic paralogs of
the miR-17.about.92 cluster, one of the best characterized groups
of miRNAs in human cancer, with oncogenic function in lymphoma,
multiple myeloma, medulloblastoma, lung, and colorectal cancer.
Ventura et al., Cell, 132(5):875-86 (2008). Interestingly,
miR-106b, miR-93, and miR-20b are members of the miR-17 family; as
such their seed sequence is identical to that of miR-17, 20a, 20b,
106a, and 106b and they are predicted to have redundant function.
Concepcion et al., Cancer J., 18(3):262-7 (2012). However, the
biological implications of intra-cluster redundancy are not
entirely understood, as clustering of microRNAs with similar seed
sequences is highly conserved, which suggests that members of the
same cluster with identical seed sequences may have functional
importance.
[0133] Members of the miR-106b.about.25 and/or miR-106a.about.363
cluster appear sensitive for differentiating HPV+ from HPV- HNSCC,
however the mechanistic basis for upregulation is not entirely
clear. HPV+HNSCC and cervical cancers are characterized by
upregulated expression of distinct and larger subsets of cell cycle
and DNA replication genes and transcription of miR-106b.about.25 is
concurrent with the protein coding gene MCM7. Indeed, MCM7 is a
known RB/E2F target gene, and E2F family transcription factors may
be paramount in mediating miR-106b.about.25 and MCM7 transcription.
The MCM7 promoter has RB/E2F binding sites and mRNA expression
correlates with miR-106b expression. This is provocative since MCM7
has been proposed as a protein biomarker that distinguishes between
HPV+ and HPV- HNSCC. Thus, the miR-106b.about.25 cluster members
may be sensitive for differentiating HPV+ from HPV- HNSCC as
relatively increased expression of this cluster is highly
characteristic of HPV+ tumors.
[0134] There is also considerable evidence that miR-106b.about.25
and its paralogs can act as bona fide oncogenes (oncomirs) with
defined roles in overcoming TGF-beta mediated growth suppression,
enhancing TGF-beta signaling, cell cycle promotion, and increased
cell survival. Petrocca et al., Cancer Cell., 13(3): 272-86 (2008).
Repression of MCM7 together with miR-106b.about.25 expression is
associated with induction of p21 and PTEN in breast and prostate
cancer. In neuroblastoma, miR-17.about.92 expression induced potent
inhibition of key TGF-.beta. signaling effectors and direct
inhibition of TGF-.beta. responsive genes. Increased expression of
miR-106b.about.25 and miR-17.about.92 in retinoblastoma was also
reported, leading to the hypothesis that in the context of genetic
Rb loss, high miR-17.about.92 expression may circumvent the need
for large numbers of genetic hits required for tumorigenesis. These
results highlight the importance of context in miRNA oncogenic
function and suggest the hypothesis that the current finding of
miR-93, miR-106b, miR-20b, and miR-363 overexpression in OPSCC
tumors supports the oncogenic activity instigated by the main viral
oncoproteins, E6, E7, and E5.
[0135] In addition to the miR106b.about.25 cluster, strong
upregulation of miR-9 is also observed in HPV+OPSCC. miR-9 has been
linked to metastatic potential via modulation of E-cadherin, acting
to prime breast cancer cells for an epithelial-mesenchymal
transition (EMT) and stimulate angiognesis. Because lymph node
metastasis is extremely common in patients with HPV+OPSCC, and
increased miR-9 levels in HPV+ relative to HPV- OPSCC were observed
in the current study and others (Gao et al., Cancer, 119: 72-80
(2013)), we asked if there was a relationship between miR-9 and
Ecadherin expression in OPSCC using qRT-PCR and western blotting of
OPSCC cell lines of known HPV; however no relationship between
miR-9 and Ecadherin was observed. Expression of E-cadherin is
generally inversely correlated to prognosis in HNSCC, with early
studies showing reduced E-cadherin as an independent prognostic
marker for metastasis and local recurrence. A recent study (n=152)
reported that the expression of E-cadherin is extensive in OPSCC
with no correlation between E-cadherin and HPV, nodal or distant
metastasis, histopathological type, or survival. Ukpo et al., Head
Neck Pathol., 6(1): 38-47 (2012). A second analysis (n=102)
suggested that HPV+/p16+ tumors from the oropharynx express high
E-cadherin and .beta.-catenin. Rampias et al., Ann Oncol., 24(8):
2124-31 (2013).
[0136] Together these results indicate that high miR-9 in HPV+OPSCC
is not directly altering Ecadherin expression, suggesting that
other functionally important miR-9 targets should be explored.
Interestingly, miR-9 may modulate the microenvironment in
HPV+OPSCC, as there is compelling evidence that miR-9 is packaged
into microvesicles or may function as a tumor suppressor. Zhuang et
al., EMBO J 31: 3513-3523 (2012). Physiological miR-9 expression
may temper innate immune responses. In cancer, there is limited
evidence that miR-9 may be involved in modulating immunoregulatory
genes, including MHC Class I and interferon regulated genes. Data
surrounding miR-9 and adaptive immune response is also lacking.
Nonetheless, the biology associated with adaptive immune response
in HPV+OPSCC is of particular interest and clinically relevant as
these tumors are being considered for immune-checkpoint inhibitor
therapy with anti-PD-1 or PD-L1 antibodies as well as adoptive cell
transfer therapy. It has also been shown that the majority of
transcriptionally active HPV+OPSCC tumors express PD-L1 (also
called B7-H1), although the significance of this and its relevance
to patient survival may be limited. Lyford-Pike et al., Cancer
Res., 73(6): 1733-41 (2013). Thus, in light of strong upregulation
of miR-9 in HPV+OPSCC, future studies will correlate miR-9
expression and the presence of immune infiltrates.
[0137] Downregulated miRNAs.
[0138] Downregulation of miR-143, miR-145, mir-199a-3p,
miR-199b.about.3p, miR-199b.about.5p, and miR-126 has been observed
in HPV+OPSCC and cervical disease. Lajer et al., Br J Cancer,
106:1526-1534 (2012). Similar results were obtained in an
organotypic keratinocyte raft culture model, showing significant
downregulation of miR-199a-5p and miR-145, suggesting a potential
mechanistic link to early stages of the HPV life cycle. Our
analyses on non-pre-amplified patient samples offered preliminary
suggestions that miR-199 and miR-145 were also downregulated in
patient samples (data not shown). After analyzing amplified
samples, and calculating relative expression of miRNAs based on the
geNorm algorithm, an extremely robust and informatics intensive
method for analyzing q-RT-PCR data, our dataset confidently
supports an HPVspecific downregulation of miR-145. The seed
sequence of miR-145 is present in the E1 ORF of a number of
papillomaviruses and E1 has been shown to be a bona fide miR-145
reactive element. Moreover, miR-145 plays a role in modulating the
viral life cycle, with a differentiation-dependent reduction in
levels of this miRNA in HPV transfected compared to control
keratinocyte raft cultures that appears to be dependent on the
function of viral E7 protein. Lentiviral forced expression of
miR-145 plays a role in controlling genome amplification with a
significant reduction of episomal viral DNA in undifferentiated
cells. These in vitro data should be interpreted as having true
biological significance, since other studies evaluating miRNA
profiles in HNSCC and cervical cancer corroborate an HPV-associated
down regulation of miR-145 and miR-143. Lajer et al., Br J Cancer,
104(5): 830-40 (2011). Taken together, these data indicate that HPV
may downregulate miR-145 in order to allow for
differentiation-specific genome amplification, and that this miRNA
remains repressed in human tumors. In this context, we can
understand miR-145 as a putative tumor suppressive miRNA in HPV
disease.
Conclusions
[0139] The overwhelming majority of HPV+HNSCC tumors arise deep
within the crypts of the tonsils of the oropharynx. These tumors
are often obscured from routine gross visualization in dental exams
and, as such, most patients present with lymph node metastases.
However, relative to patients with HPV- OPSCC, HPV+ status is
positively correlated with a 2-3-fold increase in overall survival,
indicating that the time is rapidly approaching whereupon HPV
status will dictate therapy. The HPV-associated oncogenic miRNA
panel identified herein may be incorporated into a multi-target
diagnostic platform that can contribute to early detection and/or
disease stratification to aid in differentiating oropharyngeal
tumors with different prognoses and thus distinct management
strategies. Furthermore, the oncogenic miRNA panel will facilitate
mechanistic elucidation of molecular factors that contribute to
OPSCC development, progression and response to therapy.
[0140] The complete disclosure of all patents, patent applications,
and publications, and electronically available material cited
herein are incorporated by reference. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
Sequence CWU 1
1
29123RNAHomo sapiens 1ucuuugguua ucuagcugua uga 23223RNAHomo
sapiens 2caaagugcuc auagugcagg uag 23322RNAHomo sapiens 3cauugcacuu
gucucggucu ga 22423RNAHomo sapiens 4caaagugcug uucgugcagg uag
23521RNAHomo sapiens 5uaaagugcug acagugcaga u 21621RNAHomo sapiens
6agcuacaucu ggcuacuggg u 21722RNAHomo sapiens 7aaaagcuggg
uugagagggc ga 22823RNAHomo sapiens 8cgcauccccu agggcauugg ugu
23922RNAHomo sapiens 9aauugcacgg uauccaucug ua 221022RNAHomo
sapiens 10ucguaccgug aguaauaaug cg 221121RNAHomo sapiens
11ugagaugaag cacuguagcu c 211223RNAHomo sapiens 12guccaguuuu
cccaggaauc ccu 231322RNAHomo sapiens 13acaguagucu gcacauuggu ua
221423RNAArtificial SequencemiR-9-5p primer 14ucauacagcu agauaaccaa
aga 231522RNAArtificial SequencemiR-20b-5p primer 15cuaccugcac
uaugagcauu ug 221622RNAArtificial SequencemiR-25-3p primer
16ucagaccgag acaagugcaa ug 221723RNAArtificial SequencemiR-93-5p
primer 17cuaccugcac gaacagcacu uug 231821RNAArtificial
SequencemiR-106b-5p primer 18aucugcacug ucagcacuuu a
211921RNAArtificial SequencemiR-222-3p primer 19acccaguagc
cagauguagc u 212022RNAArtificial SequencemiR-320a primer
20ucgcccucuc aacccagcuu uu 222123RNAArtificial SequencemiR-324-5p
primer 21acaccaaugc ccuaggggau gcg 232222RNAArtificial
SequencemiR-363-3p primer 22uacagaugga uaccgugcaa uu
222322RNAArtificial SequencemiR-126-3p primer 23cgcauuauua
cucacgguac ga 222421RNAArtificial SequencemiR-143-3p primer
24gagcuacagu gcuucaucuc a 212523RNAArtificial SequencemiR-145 5p
primer 25agggauuccu gggaaaacug gac 232622RNAArtificial
SequencemiR-100a-3p primer 26uaaccaaugu gcagacuacu gu
222723DNAArtificial SequenceLNA probe 27tcatacagct agataaccaa aga
232821DNAArtificial SequenceLNA probe 28atctgcactg tcagcacttt a
212923DNAArtificial SequenceLNA probe 29gaacaggtag tctgaacact ggg
23
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